Rearranged T-cell receptor beta genes represent powerful targets for quantification of minimal residual disease in childhood and adult T-cell acute lymphoblastic leukemia

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Abstract

Current MRD studies in T-cell acute lymphoblastic leukemia (T-ALL) mainly use T-cell receptor gamma, delta and SIL-TAL1 gene rearrangements as MRD-PCR targets. However, low frequency or limited diversity of these markers restricts the number of evaluable patients, particularly because two markers are recommended for MRD monitoring. Hence, we developed a new strategy implementing the TCR beta (TCRB) locus for MRD quantification. The frequency and characteristics of complete and incomplete TCRB rearrangements were investigated in 53 childhood and 100 adult T-ALL patients using the BIOMED-2 multiplex PCR assay. Clonal rearrangements were identified in 92% both childhood and adult T-ALL (Vβ–Dβ–Jβ rearrangements in 80%, Dβ–Jβ rearrangements in 53%). Comparative sequence analysis of 203 TCRB recombinations revealed preferential usage of the ‘end-stage’ segment Jβ2.7 in childhood T-ALL (27%), whereas Jβ2.3 was most frequently involved in adult T-ALL (24%). In complete rearrangements, three downstream Vβ segments (19–1/20–1/21–1) were preferentially used. Subsequently, a TCRB real-time quantitative PCR assay to quantify MRD with 13 germline Jβ primer/probe combinations and allele-specific oligonucleotides was developed and applied to 60 clonal TCRB rearrangements. The assay allowed the detection of one leukemic cell within at least 104 polyclonal cells in 93% of cases and will be of high value for future MRD studies.

Introduction

Although modern treatment induces complete remission in more than 80% of patients with T-cell acute lymphoblastic leukemia (T-ALL),1,2 most patients still harbor substantial numbers of leukemia cells after induction. Despite intensive postremission therapy many T-ALL patients relapse, mainly during or shortly after the end of therapy.1,2 Individualization of ALL treatment might improve the outcome and long-term quality of life. This may be achieved through the detection and quantification of subclinical levels of residual leukemia (MRD), thereby providing important information on the effectiveness of treatment and the risk of an impending relapse. Several studies, mainly in childhood ALL, have shown that MRD is an independent prognostic factor3,4,5,6,7,8,9 and sequential and sensitive MRD quantification allows a precise risk group classification.8,9 In this context, significant differences in the kinetics of MRD have been reported to occur in T-ALL patients compared to those with B-cell precursor ALL,6,7,9 although only few MRD studies focus on T-ALL.

A technique that allows a simple, precise and sensitive quantification of MRD in ALL is the real-time quantitative PCR (RQ-PCR) of clonally rearranged immune genes.10,11,12,13,14,15,16

So far, mainly clonal T-cell receptor gamma (TCRG) and delta (TCRD) gene rearrangements as well as SIL-TAL1 fusion genes have been used as PCR tools for MRD quantification in T-ALL.1,2,3,8,17,18,19,20,21 However, the SIL-TAL1 fusion genes are detected in only 5–25% of T-ALL patients,1,22 and also clonal TCRD gene rearrangements are found in just about 50% of patients.23,24 In addition, crosslineage immunoglobulin heavy-chain rearrangements can serve as clonal markers in T-ALL, but they are also relatively rare.25 In contrast, the vast majority of T-ALL demonstrates TCRG gene rearrangements, but quantitative PCR approaches often do not result in satisfactory sensitivity and specificity due to the limited junctional and combinatorial diversity of this locus.13 As the use of at least two clonal immune gene markers is recommended for PCR-mediated MRD monitoring,2,26,27,28 additional PCR targets for MRD quantification in T-ALL are highly warranted.

Clonal TCR beta (TCRB) gene rearrangements theoretically represent ideal MRD targets because the extensive combinatorial repertoire of TCRB rearrangements and their large hypervariable region should allow a highly sensitive and specific detection of clinically occult residual tumor cells by clone-specific PCR. Furthermore, TCRB genes are rearranged in more than 90% of all T-ALL and therefore might be applicable as sensitive MRD-PCR target in most T-ALL patients.24,29,30

However, the extensive germline-encoded TCRB repertoire renders PCR assays for clonality assessment difficult. Attempts to develop DNA-based PCR methods for the detection of clonal TCRB gene rearrangements included the use of highly degenerate consensus primers31,32 or application of the time-consuming multiple tube assay.33,34 As these approaches have their limitations in applicability or detection rate, TCRB rearrangements have rarely been used for MRD assessment.

Within the scope of the BIOMED-2 Concerted Action ‘PCR-based clonality studies for early diagnosis of lymphoproliferative disorders, we developed a new DNA-based multiplex TCRB PCR approach by design of multiple Vβ and Jβ primers that cover virtually all functional Vβ, Dβ and Jβ gene segments.35 The assay is applicable for GeneScanning and heteroduplex analysis of the PCR products and also detects the incomplete TCRB rearrangements with a total of three multiplex PCR reactions.35 This approach allows a simple screening of T-ALL samples for the presence of clonal TCRB gene rearrangements and facilitates the use of TCRB as a target for MRD assessment.

In this report, we applied the BIOMED-2 multiplex TCRB PCR assay for the identification of clonal TCRB gene rearrangements in a large series of pediatric and adult T-ALL patients. Extensive sequence analyses allowed comparative evaluation of genotypic characteristics of clonal TCRB gene rearrangements. Subsequently, we developed a new RQ-PCR assay for the detection of TCRB gene rearrangements using a limited number of germline Jβ TaqMan probes and reverse primers in combination with allele-specific oligonucleotides (ASO). Our data indicate that TCRB gene rearrangements represent an excellent PCR target for sensitive and specific quantification of residual leukemia cells in T-ALL.

Patients, materials and methods

Patient samples

Cell samples were obtained from 153 T-ALL patients (100 adults and 53 children) at initial diagnosis. We also studied several BM follow-up samples of four adult and four childhood patients who were treated according to the German multicenter ALL Study (GMALL) 05/93 or 06/99 protocol or the Dutch Childhood Leukemia Study Group ALL-8 protocol.

Mononuclear cells (MNCs) were isolated from all samples and stored at −80°C before DNA extraction. Pooled MNC DNA from equivalent amounts of five healthy donors served as a polyclonal control. DNA was isolated by standard procedures and quantified by spectrometric analysis.

Identification of clonal TCRB rearrangements

The BIOMED-2 multiplex TCRB PCR assay (with 23 consensus Vβ, two Dβ and 13 Jβ primers; InVivoScribe, Calsbad, CA, USA) was used for the identification of clonal TCRB rearrangements.35 Vβ primer codes were derived from their corresponding Vβ segments using the nomenclature that was proposed by Arden et al.36 Appropriate positive and negative controls were included in all experiments.

Reaction products were analyzed by heteroduplex analysis as described previously.37

Direct sequencing

Clonal PCR products of most T-ALL patients were directly sequenced. The dominant (homoduplex) band within the expected size range was excised and eluted from the polyacrylamide gel and sequenced. Sequence reactions were carried out in both directions, using either the complete set of Vβ primers or the Jβ Tube A or Jβ Tube B primers in a multiplex approach. In case of ambiguous sequence information concerning the NDN, but identifiable Vβ or Jβ region, sequence reaction was repeated subjecting only the involved Vβ or Jβ primer to the sequencing reaction. For the analysis of a single incomplete TCRB rearrangement, it was also possible to subject an aliquot of the unpurged PCR product directly to cycle sequencing reaction using the involved Dβ primer.

For sequencing, the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Weiterstadt, Germany) was used and the reaction products were analyzed on the ABI PRISM 377 automated sequencer (see, for details, Linke et al38). For sequencing of the sense strand of complete TCRB rearrangements 1 pmol of each Vβ primer was added and for the antisense strand 10 pmol of each Jβ primer. For incomplete rearrangements 10 pmol of the involved Dβ primer was put into the reaction. Vβ, Dβ and Jβ gene segments involved in the recombinations were identified in comparison with published sequences, using the ImMunoGeneTics database (IMGT; http://imgt.cines.fr, initiator and coordinator: Marie-Paule Lefranc, Montpellier, France).39 Vβ gene designation according to Rowen et al40 was applied. For correspondence between this nomenclature and Vβ primer names derived from designation according to Arden et al36 (see Figure 1).

Figure 1
figure1

Profiles of TCRB Vβ gene segment usage in adult (79 alleles) and childhood (47 alleles) patients with T-ALL as determined by PCR and direct sequence analysis. X-axis: arrangement of TCRB Vβ segments according to germline order. Line (A) Gene segment nomenclature according to Arden et al,36 line (B) Vβ nomenclature as proposed by Rowen et al40 and adopted by IMGT.39

RQ-PCR primers and TaqMan probes

As described for other immune gene rearrangements,10,11,13,14,16,21,41 a set of germline TCRB Jβ primers and TaqMan probes (Jβ1.1–1.6 and Jβ2.1–2.7) was constructed according to published Jβ sequences.40,42 The sequences of the oligonucleotides are given in Figure 2. In addition, ASO primers were created individually for every single patient complementary to the sequence of the TCRB N–Dβ–N junctional region using Primer Express 1.0 software (Applied Biosystems) and OLIGO 6.3 software (W Rychlik, Molecular Biology Insights, Inc., Cascado, CO, USA).

Figure 2
figure2

Schematic representation of reverse primers (R) and probes (T) for TaqMan-based RQ-PCR analysis of TCRB gene rearrangements. 5′ position of the primers and probes downstream (+) relative to the RSS of the gene segment is shown as well as each primer/probe sequence.

RQ-PCR

RQ-PCR was performed on an ABI PRISM 7700 thermal cycler (Applied Biosystems) as described elsewhere.10 Data were collected and analyzed using the Sequence Detection Software (Applied Biosystems).

Standards were generated from cell samples collected at initial diagnosis that were serially diluted in pooled polyclonal DNA to give final concentrations of (100), 10−1, 10−2, 10−3, (5 × 10−4), 10−4, (5 × 10−5), 10−5, 10−6 (dilution steps in parenthesis were not always included). Experiments were generally performed in two- to four-fold. Specificity was tested by parallel amplification of normal polyclonal MNC DNA (tested in two- to six-fold).

The sensitivity was defined as the last dilution step generating a positive signal in the absence of a signal from polyclonal control DNA. In case of nonspecific amplification, cycle threshold (CT) values of the last dilution step with a specific amplification product had to be at least one cycle lower than the lowest CT value found in control MNC DNA (tested in two- to six-fold). The dilution steps yielding quantitative data with respect to the number of allele-specific DNA copies (reproducible range) were defined as follows: (1) maximal difference in CT values of 2.0 between two- to four-fold repetitions. (2) In case of nonspecific amplification, the CT value of the dilution step defining the lower limit of the reproducible range for quantification had to be at least three cycles lower than the CT values of polyclonal control DNA. (3) The standard curve within this reproducible range should have a correlation coefficient of at least 0.95 with (4) a slope between 3.1 and 3.9.

Before analysis of follow-up samples, each RQ-PCR assay was optimized for the highest sensitivity and specificity in testing different annealing temperatures. To confirm DNA integrity and to normalize for differences in amount and quality of DNA samples, the albumin gene was used as an internal reference as described previously.10 The calculation of MRD levels was based on comparative CT analysis between follow-up samples and standards, MRD values were specified in relation to target copy number in the diagnostic sample.

Results

Frequency of TCRB gene rearrangements in T-ALL

DNA samples of 153 patients (100 adults and 53 children) with T-ALL were studied by TCRB-PCR to identify clonal rearrangements of the TCRB gene. Clonal TCRB gene rearrangements were found in 92% (140/153) of the patients (both adults and children). Frequencies of the different types of rearrangements are summarized in Table 1. In adult T-ALL, complete TCRB Vβ–Dβ–Jβ recombinations were found in 81% (81/100) of patients and 52% (52/100) of cases demonstrated incomplete Dβ–Jβ joinings. In more than half of the cases (59%) at least two rearrangements were detected, whereas in 33 out of 100 (33%) only one rearrangement was observed (25 complete and eight incomplete rearrangements). As incomplete rearrangements of the Jβ2 region can occur on the same allele as Jβ1 rearrangements, it is impossible to assess the allelic TCRB gene configuration by PCR analysis only.

Table 1 TCRB gene rearrangement patterns in T-ALL as detected by PCR analysis

In pediatric T-ALL, a total of 52 complete (Vβ–Dβ–Jβ) and 37 incomplete (Dβ–Jβ) TCRB gene rearrangements were found in 77% (41/53) and 55% (29/53) of patients, respectively. Comparable with adult T-ALL, in the majority of childhood T-ALL (31/53, 58%) at least two TCRB gene rearrangements were detected. Samples of 17 patients (32%) revealed one clonal TCRB gene rearrangement, including 12 complete and five incomplete rearrangements (Table 1).

In summary, in both adult and childhood T-ALL clonal TCRB gene rearrangements were detected with the BIOMED-2 TCRB primer set in more than 90% of patients.

In 63 immunophenotyped T-ALL, all sCD3+/TCR (6/6) and sCD3+/TCRγδ+ (2/2) cases demonstrated complete Vβ–Jβ rearrangements, compared to 36 out of 47 sCD3 T-ALL (77%); sCD3+/TCR and sCD3 cases were partially biallelic and were associated with additional Dβ–Jβ joinings in 45% (19/42) of cases. In addition, six out of eight sCD3+/TCRαβ+ T-ALL demonstrated clonal Vβ–Jβ rearrangements by PCR, the remaining two cases probably corresponding to a failure of the BIOMED-2 TCRB PCR. All complete TCRB gene rearrangements in sCD3+/TCRαβ+ turned out to be functional (ie were in frame without stop codons) on at least one allele. The other immunophenotypic subtypes were more likely to have undergone solely nonfunctional Vβ–Jβ rearrangements: 16/36 sCD3, 1/6 sCD3+/TCR and 2/2 sCD3+/TCRγδ+ T-ALL demonstrated exclusively complete TCRB rearrangements with stop codons or frameshifts.

TCRB gene segment usage in T-ALL

A total of 203 clonal TCRB junctional regions were sequenced: 126 complete Vβ–Jβ rearrangements (47 generated from pediatric and 79 from adult T-ALL) and 77 incomplete Dβ–Jβ rearrangements (37 pediatric and 40 adult T-ALL sequences).

Sequence analysis of 126 different complete Vβ–(Dβ)–Jβ recombinations allowed the identification of 41 different Vβ segments. Their frequencies are summarized in Figure 1. TCRBV20-1 was most prominent in both adult and childhood T-ALL (11 and 9%). TCRBV19 and 21-1 were also common in both subgroups. Vβ segments TCRBV5-1 and 18 occurred more frequently in adult T-ALL, whereas segments 2-1, 5-3 and 7-9 were more frequent in childhood T-ALL.

A Dβ segment within complete TCRB rearrangements was detected in 70% (adults) and 55% (children), respectively (Table 2). In adult T-ALL, Dβ1 predominated over Dβ2 (43 vs 27%), whereas in pediatric T-ALL usage of Dβ1 and Dβ2 segments occurred with the same frequency (28% both). In incomplete Dβ–Jβ rearrangements Dβ2 was preferentially used in both patient groups.

Table 2 Frequency of Dβ and Jβ usage in TCRB gene rearrangements in childhood and adult T-ALL

Frequencies of different Jβ family members are shown in Table 2. Predominance of TCRBJ2 rearrangements was characteristic for adult (TCRBJ2/TCRBJ1 ratio of 2.2) and pediatric T-ALL (ratio 2.4). However, discrepancies between the two subgroups were observed when complete and incomplete rearrangements were considered separately. In pediatric T-ALL, a relative increase in Vβ–Jβ1 rearrangements and Dβ–Jβ2 rearrangements was observed in comparison to adult T-ALL, although this difference did not reach statistical significance (P>0.05 by χ2 test). Concerning Jβ usage, the most common Jβ segment was Jβ2.3 in adults and the ‘end-stage’ segment Jβ2.7 in children, while no Jβ2.4 or Jβ2.6 segments were identified in this series (see, for details, Table 2).

Junctional regions of TCRB rearrangements in T-ALL

Table 3 summarizes the characteristics of the junctional region sequences of TCRB rearrangements in pediatric and adult T-ALL. Results were largely comparable between these two patient groups. The sizes of the junctional NDN region in complete Vβ–Dβ–Jβ rearrangements differed from 0 to maximally 47 nucleotides with an average of 18.0. Average length of the interposed Dβ segment was 7.2 nucleotides, the number of deletions and insertions was comparable between adult and childhood T-ALL (see, for details, Table 3). The junctional region of incomplete Dβ–Jβ recombinations contained a mean number of 7.2 inserted nucleotides (range 0–22). Trimming at the ends of the Dβ and Jβ gene segments resulted in the deletion of 9.7 (adults) and 10.8 nucleotides (children), equally distributed over Dβ and Jβ.

Table 3 Characteristics of TCRB junctional regions in childhood and adult T-ALL

RQ-PCR analysis

The applicability of the primer/probe set for RQ-PCR was tested in combination with ASO forward primers for 60 TCRB gene rearrangements (48 complete and 12 incomplete rearrangements, Table 4). A sensitivity of 10−3 was reached for all 60 rearrangements and 10−4 for 56 (93%) of the 60 investigated rearrangements (Tables 4). The standard annealing temperature was 59–60°C, but due to nonspecific amplification of polyclonal control MNC DNA the temperature had to be increased to 61–69°C in 23 (38%) cases. This was particularly necessary for incomplete TCRB rearrangements (eight out of 12 cases). For complete TCRB rearrangements, elevated annealing temperatures had to be used in 15 out of 48 cases. Despite optimization, the sensitivity did not exceed 10−3 in two and 5 × 10−4 in another two cases. Reasons were high background (2/4 rearrangements) and limited specific amplification (2/4). In case A7115, increasing the annealing temperature led to the disappearance of background and to limited specific amplification, resulting in a sensitivity of 5 × 10−4.

Table 4 RQ-PCR analysis of (a) complete (Vβ–Dβ–Jβ) and (b) incomplete (Dβ–Jβ) TCRB gene rearrangements in T-ALL patients

Eight additional cases did not fulfill the strict criteria of a reproducible range up to 10−4 (limit 10−3: one Vβ–Jβ rearrangement; limit 5 × 10−4: five Vβ-Jβ and two Dβ–Jβ rearrangements). Two problems led to a limited range of quantitative analysis: (1) in three cases normal MNC resulted in amplification with a CT within three cycles from the specific amplification product despite the optimization of annealing temperature. (2) Maximal difference in CT values of two- to four-fold repetitions of the defining dilution step exceeded 2.0 in another five cases.

We tried to identify parameters of TCRB junctional regions, which led to good sensitivities in RQ-PCR. There was no apparent correlation between the reproducible range and Vβ gene segments used. However, Jβ segments Jβ2.3, Jβ2.5 and Jβ2.7 dominated in TCRB gene rearrangements leading to reproducible ranges >10−4 (11/12, 92% rearrangements), whereas these Jβ gene segments were involved in only 24/48 (50%) of cases with a sensitive reproducible range up to 10−4 (P<0.05 by χ2 test). No significant dependency on the number of inserted or deleted nucleotides was found. Solely, the number of insertions in complete Vβ–Dβ–Jβ recombinations showed a tendency to be higher in recombinations with a higher reproducible range (mean 19.1 vs 13.0).

TCRB gene rearrangements can be selected as targets for MRD with a high chance to reach a sensitivity of at least 10−4 (96% of complete and 84% of incomplete TCRB rearrangements) and a reproducible range up to 10−4 in 80% of the cases (40 of 48 (83%) complete and eight of 12 (67%) incomplete TCRB rearrangements).

Quantitation of MRD in follow-up samples

To test the usefulness of the new TCRB RQ-PCR assay for MRD studies in clinical cases, MRD was quantified in a total of 74 follow-up samples of four pediatric and four adult T-ALL with a total of nine different TCRB targets. MRD levels in the follow-up samples from six patients were compared to the results with previously obtained data using other MRD-PCR targets.

Patients P5158, P0807, A6033 and A11039 relapsed after achieving a complete remission (Figure 3). In patients P5158 and P0807, leukemic cells were detected in all follow-up samples until relapse. Patients A6033 and A11039 apparently had an initial decrease in tumor burden, but MRD was intermittently detectable during therapy and showed a quantitative increase prior to clinical relapse. Comparison of MRD values using two different targets was possible for three of these patients and showed comparable results in two cases. However, in patient P5158 differences in corresponding MRD values were seen. While TCRB and TCRD MRD values in week 3 were almost identical, MRD data in week 22 and at time of clinical relapse (week 50) diverged: values obtained by TCRB RQ-PCR were significantly lower than corresponding TCRD RQ-PCR values. This patient was analyzed in detail for the configuration of the involved TCRB, TCRG and TCRD genes at diagnosis and relapse. An alteration in TCRB gene rearrangements between diagnosis and relapse was seen, whereas the TCRG and TCRD rearrangements were stable.28

Figure 3
figure3

Applicability of RQ-PCR analysis of TCRB gene rearrangements for MRD detection in follow-up samples of eight patients with T-ALL. RQ-PCR results using clonal TCRB gene rearrangements as targets (squares) were compared to MRD values obtained by RQ-PCR of TCRG or TCRD gene rearrangements (triangles). In patient P5158, the TCRB rearrangement was concluded to be instable (based on mixed heteroduplex analysis of the diagnosis and relapse sample), but appeared to be present in a subclone at relapse as detected by RQ-PCR analysis. RQ-PCR pos<RR: RQ-PCR positive, but value below reproducible range; neg: negative (no specific amplification).

Patients P5471, P5638, A6526 and A6768 remained in remission. The MRD levels in these four patients showed a rapid decrease and subsequently dropped to levels undetectable by both targets. Minor discrepancies between corresponding MRD values were seen in some samples with residual leukemia close to the detection limit of the assays. In all these cases, MRD was discovered only by one of the two targets at levels below the reproducible range, reflecting differences in the sensitivity of the assays each time (patient P5471, A6526 and A6768: TCRB gene rearrangement represents the more sensitive target; patient P5638: TCRG assay is more sensitive, Figure 3).

Discussion

TCRB gene rearrangements are of great interest for clonality assessment and follow-up studies in T-cell malignancies, because their extensive combinatorial repertoire and large hypervariable region theoretically enables a highly specific and sensitive detection of malignant cells. However, the large germline-encoded TCRB repertoire renders PCR assays more difficult for this locus compared to TCRG and TCRD PCR approaches. We recently developed a new and convenient TCRB PCR assay via the BIOMED-2 Concerted Action,35 which enabled us to develop a simple strategy to detect and sequence clonal TCRB gene rearrangements at the DNA level and to quantify MRD in T-ALL by RQ-PCR analysis. The application of the BIOMED-2 assay in a large series of 100 adult and 53 childhood T-ALL patients revealed the presence of at least one clonal TCRB gene rearrangement in more than 90% of adult (92/100) and childhood (48/53) T-ALL. However, two of the PCR-negative cases were TCRαβ+ T-ALL, which should demonstrate at least one complete clonal TCRB gene rearrangement.30 These two cases probably correspond to a failure of the BIOMED TCRB PCR. As expected, all complete TCRB gene rearrangements in the remaining sCD3+/TCRαβ+ cases turned out to be functional on at least one allele. The other immunophenotypic subtypes were less likely to have undergone functional Vβ–Jβ rearrangements: 16/36 sCD3, 1/6 sCD3+/TCR and 2/2 sCD3+/TCRγδ+ T-ALL exclusively demonstrated nonfunctional complete TCRB rearrangements. However, the number of immunophenotyped cases was too small to allow firm conclusions. Asnafi et al43 subdivided a large series of T-ALL on the basis of sCD3, TCRαβ, TCRγδ and cTCRβ expression: absence of rearranged TCRB genes was virtually restricted to immature sCD3/cTCRβ T-ALL, in 37% of sCD3+/TCRγδ+ cases rearrangements were exclusively incomplete. Also in 4/45 cTCRβ+ and TCRαβ+ T-ALL, PCR detection of clonal Vβ–Jβ joinings failed, in these cases probably corresponding to a PCR failure.

The overall proportion of TCRB-PCR-positive cases was comparable to those previously published for T-ALL patients using Southern blot analysis: Szczepanski et al24 detected at least one clonal TCRB gene rearrangement in 18 out of 22 cases with T-ALL (82%), Sazawal et al44 in 39/45 (87%). Langerak et al30 analyzed a larger series of 156 T-ALL cases and detected at least one clonal TCRB rearrangement in 88%. Nevertheless, some discrepancies were also found in rearrangement patterns: the total percentage of alleles with complete Vβ–Jβ rearrangements (52/106 alleles in children and 109/200 alleles in adults) appeared to be somewhat lower than described previously.30 This may be caused by PCR-related problems: as the diagnostic PCR approach is a multiplex assay, theoretically one of the two clonally rearranged TCRB genes may be preferentially amplified, thereby resulting in an underestimation of the number of biallelic complete rearrangements. In addition, some nonfunctional Vβ gene segments are partially not recognized by the BIOMED-2 Vβ primer set.35 Nevertheless, as the overall percentage of PCR detected TCRB-positive T-ALL cases is comparable to those previously discovered by Southern blotting, the BIOMED-2 PCR technique offers a highly useful alternative for defining MRD targets, especially as it is much more rapid, less expensive, requires only small amounts of DNA and allows direct sequencing of clonal rearrangements.

Based on the sequence analysis of a total of 203 clonal TCRB rearrangements, we were able to establish characteristics of TCRB gene rearrangements in adult and childhood T-ALL. Firstly, rearrangements of the TCRBJ2 locus predominated in both groups (69% in adults and 70% in children), which is in accordance with earlier reports based on Southern blot analysis.24,29,30 However, the distribution of Jβ2 usage differed between complete and incomplete rearrangements: the percentage of incompletely rearranged alleles involving the Jβ2 region was higher in childhood T-ALL (Dβ–Jβ2/Dβ–Jβ1 ratio 3.1) opposed to adults (Dβ–Jβ2/Dβ–Jβ1 ratio 1.4), whereas the percentage of complete Vβ–Jβ2 rearrangements was decreased in childhood T-ALL (ratio 1.9 vs 3.0). These observations (relative increase in complete Vβ–Jβ1 and incomplete Dβ–Jβ2 rearrangements) potentially belong together: a complete Vβ–Jβ1 rearrangement still allows an additional Dβ2–Jβ2 rearrangement on the same allele, whereas no extra Dβ–Jβ rearrangement is possible on the same allele in case of a complete Vβ–Jβ2 rearrangement. Actually in our series of T-ALL, a complete Vβ–Jβ1 rearrangement was associated with a Dβ2–Jβ2 recombination in 72% of cases, whereas in case of Vβ–Jβ2 rearrangements an extra Dβ2–Jβ2 was found in only 27% of cases (P<0.05 by χ2 test). The phenomenon of age-dependent differences in the Jβ2/Jβ1 ratio correlates to a finding by Langerak et al30, who described discrepancies in the Jβ2/Jβ1 distribution depending on the immunophenotype of T-ALL: TCRαβ+ T-ALL exhibited the same differences in Jβ2/Jβ1 distribution vs immature sCD3 T-ALL as we observed in childhood T-ALL vs adult T-ALL. This analogy is potentially an additional indication of the more immature T-ALL genotype in adults compared to children.24

The pattern of Jβ segment usage was comparable to that published for peripheral blood lymphocytes in healthy individuals: Jβ1.3, Jβ1.4, Jβ2.4 and Jβ2.6, which are rarely used by normal peripheral blood T-lymphocytes45 were also infrequent (Jβ1.3 and Jβ1.4) or even absent (Jβ2.4 and Jβ2.6) in our series of T-ALL. In childhood T-ALL, the Jβ2.7 as the most downstream Jβ segment was used most frequently, whereas Jβ2.3 predominated in adult T-ALL. Theoretically, the complete absence of detectable Jβ2.4 or Jβ2.6 gene rearrangements could be a failure of the corresponding BIOMED-2 Jβ primers. However, in an ongoing investigation of B-lineage ALL, we detected clonal TCRB gene rearrangements containing both Jβ gene segments using the BIOMED-2 primer set (data not shown). In addition, every single primer was tested for its function within the scope of the BIOMED-2 Concerted Action.35

We observed a preferential usage of the more downstream Vβ segments in complete rearrangements (Figure 1). Particularly, the Vβ segments TCRBV19, TCRBV20 and TCRBV21 were present in about one-quarter of all complete rearrangements, although a total number of 41 different Vβ gene segments was involved in the 126 sequenced TCRB gene rearrangements.

Rearrangements to the most downstream Jβ2.7 gene segment do not allow ongoing rearrangements owing to Jβ replacement and therefore can be perceived as end-stage recombination events. However, about 20% of T-ALL patients demonstrate clonal evolution of TCRB gene rearrangements.28 Several clonal evolution phenomena are described, including Vβ–Jβ replacement.28 This mechanism requires initial usage of the more downstream Vβ gene segments that we found to be rearranged in the majority of complete TCRB gene rearrangements. Oligoclonality is also described as a factor of clonal instability.26,28 In our series of T-ALL, we found evidence for subclone formation at the TCRB level in one patient (A12805) due to ongoing Vβ to Dβ–Jβ joining, as sequences of an incomplete Dβ2–Jβ2.3 and a complete Vβ4.1–Dβ2–β2.3 rearrangement contained a common Dβ–Jβ stem.

The junctional region diversity was extensive with an NDN region consisting of a mean number of 18 nucleotides without differences between pediatric and adult T-ALL. Incomplete Dβ–Jβ rearrangements showed a mean of 7.2 inserted nucleotides, which turn also these junctional regions into attractive targets for ASO primers.

In previous studies, we and others already demonstrated the applicability of RQ-PCR assays using ASO primers and consensus probes for sensitive and specific detection of clonal IGH,10,11,16 IGK-Kde,14 TCRG13 and TCRD21,41 rearrangements in patients with ALL. In the present report, we developed a TCRB RQ-PCR assay employing germline Jβ region probes and reverse primers in combination with ASO forward primers (Figure 2). We demonstrate that this PCR method allows a specific, sensitive and reproducible detection of residual tumor cells over a wide range.

Owing to the lack of homology between the different Jβ segments, it was necessary to design a specific germline probe and primer for every single Jβ gene. In combination with an ASO primer, this RQ-PCR approach allowed to detect one leukemic cell in a background of at least 104 normal cells in 56 of the 60 investigated TCRB rearrangements (93%). A sensitivity of 10−3 was even reached in all cases. Exact quantification up to a level of 10−4 was possible in 48/60 cases. In contrast to TCRG rearrangements as targets for RQ-PCR-based MRD analysis,13 nonspecific amplification of normal MNC DNA was not the critical factor for inferior quantification limits. In most cases (5/12) CT variations of more than 2.0 between two- to four-fold repetitions of a dilution step limited reproducibility and thus exact quantification of MRD in follow-up samples. Larger variability for samples containing low levels of tumor DNA can be explained by an increased significance of sampling errors and stochastic effects.13 These factors limit exact MRD quantification in case of low tumor burden, but occur irrespective of the type of target used for quantification and are not associated with the risk of false positivity. Only in five out of 12 rearrangements with a quantification limit not reaching 10−4, nonspecific amplification occurred at CT values nearby the 10−4 dilution, despite optimization of the annealing temperature. All five rearrangements involved Jβ2.3, 2.5 or 2.7 segments, which were found to be frequently rearranged in peripheral blood T-lymphocytes.34,45

Using the newly developed TCRB RQ-PCR strategy, we quantified MRD in a total of 74 follow-up samples in eight T-ALL patients demonstrating concordance of MRD kinetics with clinical course. Comparative MRD analyses with two different immune gene targets led to comparable results in all but one (P5158) cases. In patient P5158, the TCRB gene rearrangement was concluded to be instable based on heteroduplex analysis of the diagnosis and relapse sample,28 but appeared to be present in a subclone at relapse by RQ-PCR analysis.

In conclusion, clonal TCRB gene rearrangements are excellent targets for RQ-PCR detection of MRD in T-ALL because of their extensive combinatorial repertoire and large junctional region. Consequently, they represent first choice PCR targets for MRD studies in T-ALL patients. However, clonal evolution can occur, which might be related to the type of TCRB rearrangement and the type of T-ALL.28

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Acknowledgements

We gratefully acknowledge Petra Chall, Petra Neumann, Frauke Hemken and Heidrun Seppelt for their technical assistance, Margot Ulrich for preparation of the figures, Sabine Hug and Regina Reutzel for providing clinical data and the German Multicenter ALL Study Group and the Dutch Childhood Leukemia Group for kindly providing the ALL samples. This work was supported by the Wilhelm Sander-Stiftung (Grant 2001.074.1), the German Compentence Network ‘Akute und Chronische Leukämien’ and by the Dutch Cancer Society/Koningin Wilhelmina Fonds (Grant SNWLK 97-1567 and Grant SNWLK 2000-2268).

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Correspondence to M Brüggemann.

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Keywords

  • TCRB genes
  • RQ-PCR analysis
  • T-ALL
  • MRD

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